Project summary We propose to advance both the resolution and sensitivity of intact protein separations for nanospray and microbore liquid chromatography-mass spectrometry. These are urgently needed to advance the tool of top-down-proteomics, which is an emerging technology that is vital for applications where multiple post-translational modifications on a single protein matter to the understanding of human health. Higher resolution is proposed through two primary innovations: the use of use of submicrometer and the design of polymer bonded phases tailored for protein separations. The proposed higher resolution of the chromatography will take some of the burden off the mass spectrometer, allowing wider use of top-down proteomics with sometimes less expensive mass spectrometers. The chromatographic separations will be developed in the context of an exciting and significant area of research in human health: epigenetics, where histone variants and post-translation modifications need to be characterized for a wide variety of diseases. The proposal outlines in the first aim how a 10X higher sensitivity has already been attained through our use of 0.5 m particles in reversed phase nanoLCMS, and describes how at least a three-fold higher resolution can be attained for difficult proteins, such as the highly cationic histones, without lowering the sensitivity. Capillaries for higher resolution in weak cation exchange chromatography and for hydrophobic interaction chromatography will be developed in the second aim for providing a second, orthogonal dimension to the separation. These will be evaluated in 2D LCMS in the third aim. Success of the proposed work will broadly impact top-down proteomics and specifically advance the field of epigenetics.